Role of cell cycle dynamics in early cell fate decisions of interneuron precursors in the MGE

抑制性中间神经元是一个小而重要的神经元群体，在功能、形态和放电模式上表现出极大的多样性。事实上，多种人类疾病，如癫痫、自闭症谱系障碍或精神分裂症，都与中间神经元功能障碍有关。根据小白蛋白（PV）或生长抑素（SST）的表达划分，两个主要的中间神经元亚群起源于内侧神经节隆起（MGE）的瞬时结构。在MGE有丝分裂祖细胞中，细胞成为PV或SST中间神经元的命运决定很早就开始了，其规范可能受到细胞分裂调节因子的影响。在这个项目中，我的目标是阐明PV和SST命运决定背后的分子程序，并探索细胞周期和分裂模式的关键调节因子如何作用于中间神经元规范。我将使用单细胞RNA测序（scRNAseq）来研究在胚胎(E)日E11.5, E13.5和E15.5指导MGE祖细胞分化的分子程序，辅助突变小鼠系。分割缺陷3 (Pard3cKO) MGE的条件敲除增加了成人前脑的SST中间神经元，而细胞周期蛋白D2敲除（cD2KO）显示PV中间神经元密度降低。E13.5的scnaseq中试实验表明，在野生型（WT） MGE中，不同类型的细胞可以被明确地识别出来，并分为9个细胞簇，分别代表放射状胶质细胞（RGCs）、中间祖细胞（IPCs）和早期神经元。与WT相比，cD2KO小鼠缺乏IPC簇，这支持了先前的文献，认为PV中间神经元起源于室下区IPC。在Pard3cKO MGE中，缺少一个RGC集群，这表明在E13.5，祖细胞过早地停止分裂以采用SST身份，这与SST神经元直接来自RGC的数据一致。E11.5和E15.5的数据将测试这些基因操作是否会改变中间神经元从MGE输出的时间，并将有助于确定关键的命运决定因素。先前对皮层的研究表明，非对称分裂和对称分裂是调节胚胎生发区神经发生和细胞数量输出的关键。然而，这些分裂模式是如何在分子水平上驱动细胞周期的，我们知之甚少。par3促进RGCs的不对称分裂，而cD2主要在IPCs中的表达表明其促进对称分裂的机制尚不清楚。与cD2相反，cD1仅在分裂的RGCs中表达，这表明cD1对不对称分裂很重要。令人兴奋的试点数据显示，PARD3与cD1或cD2的物理相互作用存在重要差异。为了探究这些生化差异的功能，我将研究与PARD3- cd1相比，PARD3和cD2的双重失活对不同胚胎年龄MGE细胞周期特征的影响，并确定成人前脑PV和SST细胞命运决定结果。

Inhibitory interneurons are a small but crucial neuronal population, which show a great diversity in function, morphology and firing pattern. Indeed, diverse human pathologies like epilepsy, autism spectrum disorders or schizophrenia are associated with interneuron dysfunctions. Two major interneuron subpopulations, classified by the expression of Parvalbumin (PV) or Somatostatin (SST), originate from the transient structure of the medial ganglionic eminence (MGE). The cell fate decision to become a PV or SST interneuron is initiated early, in MGE mitotic progenitors whose specification may be influenced by regulators of cell divisions.In this project I aim to elucidate the molecular program underlying the PV vs. SST fate decision and probe how key regulators of the cell cycle and division mode act toward interneuron specification.I will use single cell RNA sequencing (scRNAseq) to investigate molecular programs directing MGE progenitor differentiation at embryonic (E) days E11.5, E13.5, and E15.5, assisted by mutant mouse lines. The conditional knockout in MGE of partition defective 3 (Pard3cKO) increases SST interneurons in the adult forebrain, while the cyclin D2 knockout (cD2KO) shows decreased PV interneuron densities. Pilot scRNAseq experiments at E13.5 show that different cell types could be robustly identified and classified into nine cell clusters in the wildtype (WT) MGE, representing radial glia cells (RGCs), intermediate progenitor cells (IPCs), and early neurons. In contrast to WT, the cD2KO mice lack an IPC cluster, supporting previous literature suggesting PV interneurons originate in the IPCs of the subventricular zone. In Pard3cKO MGE an RGC cluster is missing, suggesting that at E13.5, progenitors prematurely stop dividing to adopt an SST identity, consistent with data that SST neurons arise directly from RGCs. Data from E11.5 and E15.5 will test whether these genetic manipulations shift the timing of interneuron output from the MGE and will help identify key fate determinants.Previous studies in cortex established that asymmetric vs. symmetric divisions are key to regulation of neurogenesis and cell number output from embryonic germinal zones. However, little is known of how these division modes are connected to driving the cell cycle at the molecular level. PARD3 facilitates asymmetric divisions of RGCs, while cD2 expression mostly in IPCs suggests that it promotes symmetric divisions through an as yet unknown mechanism. In contrast to cD2, cD1 is only expressed in dividing RGCs, suggesting cD1 is important for asymmetric divisions. Exciting pilot data show PARD3 to physically interact with cD1 or cD2 with important differences. To probe the function of these biochemical differences, I will examine the impact of dual inactivation of PARD3 and cD2, compared to PARD3-cD1, on MGE cell cycle characteristics at different embryonic ages and determine the PV vs. SST cell fate decision outcomes in the adult forebrain.